Genome-Wide Analyses of SlFWL Family Genes and Their Expression Profiles under Cold, Heat, Salt and Drought Stress in Tomato

PLAC8 is a cysteine-rich protein that serves as a central mediator of tumor evolution in mammals. PLAC8 motif-containing proteins widely distribute in fungi, algae, higher plants and animals that have been described to be implicated in fruit size, cell number and the transport of heavy metals such as cadmium or zinc. In tomatoes, FW2.2 is a PLAC8 motif-containing gene that negatively controls fruit size by regulating cell division and expansion in the carpel ovary during fruit development. However, despite FW2.2, other FWL (FW2.2-Like) genes in tomatoes have not been investigated. In this study, we identified the 21 SlFWL genes, including FW2.2, examined their expression profiles under various abiotic adversity-related conditions. The SlFWL gene structures and motif compositions are conserved, indicating that tomato SlFWL genes may have similar roles. Cis-acting element analysis revealed that the SlFWL genes may participate in light and abiotic stress responses, and they also interacted with a variety of phytohormone-responsive proteins and plant development elements. Phylogenetic analyses were performed on five additional plant species, including Arabidopsis, pepper, soybean, rice and maize, these genes were classified into five subfamilies. Based on the results of collinearity analyses, the SlFWL genes have a tighter homologous evolutionary relationship with soybean, and these orthologous FWL gene pairs might have the common ancestor. Expression profiling of SlFWL genes show that they were all responsive to abiotic stresses, each subgroup of genes exhibited a different expression trend. Our findings provide a strong foundation for investigating the function and abiotic stress responses of the SlFWL family genes.


Introduction
FW2.2 (fruit weight 2.2), the first locus identified as a significant QTL regulating tomato fruit weight, controls tomato fruit size by negatively regulating the number of cell divisions in the carpel ovary [1,2]. It plays a crucial role in the domestication and agronomic advancement of tomatoes and contributes up to 30% of the diversity in fruit weight variation, making it a vital factor in the evolution of fruit size [1]. FW2.2 is a member of a broad family of cysteine-rich eukaryotic proteins with a placenta-specific 8 conserved structure, originally discovered in mammalian placental proteins [3,4]. In tomatoes, the FW2.2 gene primarily affects fruit size by regulating the number of cell divisions and expansions in well characterized, and few studies have been reported on their response to various abiotic stresses. In this study, we characterized the SlFWL gene family in tomatoes and performed a comprehensive analysis of their protein physicochemical properties, chromosomal localization, gene structure, protein motifs, phylogenetic relationships and subcellular localization predictions. We also analyzed their expression profiles in response to abiotic stress such as low temperature (4 • C), high temperature (42 • C), salt (NaCl) and drought (PEG) stimuli. This study provides a better understanding of the structural characteristics of tomato SlFWL genes and serves as a basis for the functional validation of SlFWL genes and their role in stress tolerance in tomato plants.

Identification of the SlFWL Genes in Tomato
To identify the members of the FWL gene family in tomatoes, we conducted HMM searches using the amino acid sequences in tomato, followed by BLASTP to detect any missing SlFWL genes. We further validated the conserved structural domain, PF04749, using NCBI-CDD. In total, we identified 21 SlFWL genes in tomatoes, including FW2.2. These genes were named based on their respective chromosome numbering. The basic characteristics of these genes and their corresponding proteins were analyzed ( Table 1). The majority of SlFWL genes encode short peptides with 100-300 amino acids, while only a few contain more than 400 amino acids. Their molecular weights range from 11.07 to 57.09 kDa, their isoelectric points range from 4.48 to 9.37, and their cysteine content spans from 2.7% to 12.8%. Most of the SlFWL genes were predicted to localize in the cell membrane, except for SlFWL6, which was predicted to localize in the nucleus. SlFWL2, SlFWL3, SlFWL10, SlFWL11 and SlFWL14 were predicted to localize in both the cell membrane and nucleus. The transmembrane structure prediction results show that more than half of the SlFWL proteins have at least one transmembrane structure. The various predictions of subcellular localization and the number of transmembrane structures may indicate the functional diversity of SlFWL proteins.

Chromosomal Localization, Phylogenetic Relationships, and Gene Structures of the SlFWL Genes
To better understand the evolutionary relationships among the SlFWL genes, we generated a phylogenetic tree using the neighbor-joining (NJ) method and visualized the gene structure using TBtools (v1.09876) ( Figure 1A). The chromosomal localization analysis revealed that all SlFWL genes were tandemly and relatively evenly distributed on 10 chromosomes of tomatoes, with at least one SlFWL gene on each chromosome ( Figure 1B). The sequence similarity of the tandemly distributed SlFWL genes was not very high, as demonstrated by the phylogenetic tree and chromosomal localization, indicating that they are unlikely to have common functions despite being physically adjacent. The intron and exon architecture of the SlFWL genes show that their exons were relatively short, and their distribution traits were comparable to those of FW2.2. SlFWL9 is a pseudogene, as it lacks a coding region. Each SlFWL gene has one to seven exons, and the distribution of the coding region of most SlFWL genes was conserved. SlFWL11 and SlFWL12 only contain 1 exon, SlFWL3 and SlFWL7 have six to seven exons, and other members have a fairly consistent distribution of 3-4 exons. The diversity of the gene structure of the SlFWL family genes suggests a variety of putative biological activities. distribution traits were comparable to those of FW2.2. SlFWL9 is a pseudogene, as it lacks a coding region. Each SlFWL gene has one to seven exons, and the distribution of the coding region of most SlFWL genes was conserved. SlFWL11 and SlFWL12 only contain 1 exon, SlFWL3 and SlFWL7 have six to seven exons, and other members have a fairly consistent distribution of 3-4 exons. The diversity of the gene structure of the SlFWL family genes suggests a variety of putative biological activities.

Conserved Motif Analysis of SlFWL Genes
The SlFWL genes in tomatoes were analyzed for conserved motifs using MEME (https: //meme-suite.org/meme/tools/meme, accessed on 16 May 2022), with the identification of 10 conserved motifs ( Figure 2). With the exception of SlFWL2 and SlFWL9, most SlFWL proteins were found to have similar conserved motifs, including motifs 1, 2, 3, 4 and 5. Motifs 3, 4 and 5 were identified as conserved sequences of the PLAC8 protein. Interestingly, motifs 3 and 4 were located at the N-terminus of these SlFWL proteins, while motif 5 was located at the C-terminus. Most SlFWL proteins also had motif 10, with only SlFWL2 and SlFWL13 lacking it. These results suggest that SlFWL proteins containing the PLAC8 structural domain were conserved during evolution. Additionally, SlFWL5, SlFWL10 and SlFWL17 had motifs 6 and 7, in which motif 7 contained a conserved sequence of a zinc finger domain. Motifs 8 and 9 were only present in SlFWL11 and SlFWL12. These results imply that the SlFWL proteins might have a relatively conserved function and have diverged during evolution.
SlFWL2 and SlFWL13 lacking it. These results suggest that SlFWL proteins containing the PLAC8 structural domain were conserved during evolution. Additionally, SlFWL5, SlFWL10 and SlFWL17 had motifs 6 and 7, in which motif 7 contained a conserved sequence of a zinc finger domain. Motifs 8 and 9 were only present in SlFWL11 and SlFWL12. These results imply that the SlFWL proteins might have a relatively conserved function and have diverged during evolution.

Analysis of Cis-Regulatory Elements in SlFWL Genes
The 2 kb upstream sequences from the translation start sites of the SlFWL genes were analyzed to identify their cis-regulatory elements and investigate their potential regulatory mechanisms in plant growth, development, and abiotic stress responses ( Figure 3, Table S1). The analysis revealed that the promoter regions of the SlFWL genes contained a large number of light response elements, with a total of 278 elements identified. Moreover, most of the SlFWL genes contained phytohormone elements, such as abscisic acid response elements, jasmonic acid response elements, and salicylic acid response elements, as well as gibberellin response elements. Additionally, a total of 22 MYB binding sites were detected, which also contained seven rhythm-related elements and six low-temperature response elements. In addition, anaerobic response elements, defense stress response elements, TC-rich repeats (defense and stress), GCN4_motif (sperm endosperm), and zein metabolism regulation response elements were also detected. These findings

Analysis of Cis-Regulatory Elements in SlFWL Genes
The 2 kb upstream sequences from the translation start sites of the SlFWL genes were analyzed to identify their cis-regulatory elements and investigate their potential regulatory mechanisms in plant growth, development, and abiotic stress responses ( Figure 3, Table S1). The analysis revealed that the promoter regions of the SlFWL genes contained a large number of light response elements, with a total of 278 elements identified. Moreover, most of the SlFWL genes contained phytohormone elements, such as abscisic acid response elements, jasmonic acid response elements, and salicylic acid response elements, as well as gibberellin response elements. Additionally, a total of 22 MYB binding sites were detected, which also contained seven rhythm-related elements and six low-temperature response elements. In addition, anaerobic response elements, defense stress response elements, TC-rich repeats (defense and stress), GCN4_motif (sperm endosperm), and zein metabolism regulation response elements were also detected. These findings suggested that the SlFWL genes may participate in many complex regulatory pathways involved in plant development, metabolic processes, and stress responses.

Homology Analysis of FWL Genes from Different Species
In order to investigate the homology of FWL proteins across different plant species, FWL proteins from 68 other species were extracted from EnsemblPlant, including proteins from dicotyledonous plants such as Arabidopsis, pepper and soybean, as well as monocotyledonous plants such as rice and maize [3,10,13,15,27]. A multispecies phylogenetic tree was generated ( Figure 4, Table S2). Based on the branching of the evolutionary tree, the FWL protein family was divided into five subfamilies. All five subfamilies are present in both monocots and dicots. Subfamily E contained most AtPCR members, with SlFWL2 located on a specific subbranch in this subfamily. SlFWL5, SlFWL9, SlFWL10, SlFWL11, SlFWL12, SlFWL16, SlFWL17, SlFWL18, SlFWL19, SlFWL20 and SlFWL21 were closely related to OsPCR3, ZmCNR5, ZmCNR6, ZmCNR8, GmFWL1, AtPCR9 and AtPCR10, belonged to subfamily A. The members of subfamily B were mainly MCA, SlFWL14 and SlFWL15 were also located in this family. Tomato FW2.2 was the only FWL member in subfamily C, which also included pepper PHT91598.1 (Cell number regulator 1) protein. SlFWL1 and SlFWL8 exist in subfamily D, which also contains AtPCR1, AtPCR2, AtPCR3, OsPCR1, OsPCR4, OsPCR5 and OsPCR6. These results suggest that FWL proteins are widely existent and diverse across plant species and exhibit sequence conservation in related species during evolution. This implies that FWL proteins may perform similar biological functions among different species. suggested that the SlFWL genes may participate in many complex regulatory pathways involved in plant development, metabolic processes, and stress responses.

Homology Analysis of FWL Genes from Different Species
In order to investigate the homology of FWL proteins across different plant species, FWL proteins from 68 other species were extracted from EnsemblPlant, including proteins from dicotyledonous plants such as Arabidopsis, pepper and soybean, as well as monocotyledonous plants such as rice and maize [3,10,13,15,27]. A multispecies phylogenetic tree was generated ( Figure 4, Table S2). Based on the branching of the evolutionary tree, the FWL protein family was divided into five subfamilies. All five subfamilies are present in both monocots and dicots. Subfamily E contained most AtPCR members, with SlFWL2 located on a specific subbranch in this subfamily. SlFWL5, SlFWL9, SlFWL10, SlFWL11, SlFWL12, SlFWL16, SlFWL17, SlFWL18, SlFWL19, SlFWL20 and SlFWL21 were closely related to OsPCR3, ZmCNR5, ZmCNR6, ZmCNR8, GmFWL1, AtPCR9 and AtPCR10, belonged to subfamily A. The members of subfamily B were mainly MCA, SlFWL14 and SlFWL15 were also located in this family. Tomato FW2.2 was the only FWL member in subfamily C, which also included pepper PHT91598.1 (Cell number regulator 1) protein. SlFWL1 and SlFWL8 exist in subfamily D, which also contains AtPCR1, AtPCR2, AtPCR3, OsPCR1, OsPCR4, OsPCR5 and OsPCR6. These results suggest that FWL proteins are widely existent and diverse across plant species and exhibit sequence conservation in related species during evolution. This implies that FWL proteins may perform similar biological functions among different species.

Collinearity Analysis of SlFWL Genes with Other Species
The extension and evolution of gene families in the genome are significantly influenced by tandem and fragmental replication between gene families, and tandem replication is a key mechanism for creating new copies of genes [28]. To investigate the evolutionary relationships of FWL genes in different plant species, we performed a synteny analysis of FWL genes between the genomes of tomato, Arabidopsis, rice (Oryza indica and Oryza japonica), maize, soybean and pepper ( Figure 5, Table S3). The results show that the number of collinear gene pairs between tomato and the other species ranged from 1 to 22, suggesting that these collinear genes might originate from the same ancestor and are functionally conserved. The highest number of collinear genes was found between the tomato and soybean genomes, while maize and rice had the lowest number of collinear genes. These findings indicate that FWL genes might have undergone a complex evolutionary history. software was applied to construct the phylogenetic tree with 1000 bootstrap replicates. The FWL proteins were used to run 1000 self-replicates and build a phylogenetic tree using the neighborjoining method on the MEGA (Version 11) program. Based on branching, the FWL family proteins were classified into five subfamilies. Each subfamily is represented by a distinct color: purple for subfamily A, red for subfamily B, yellow for subfamily C, blue for subfamily D, and green for subfamily E. Sl (Solanum lycopersicum) stands for tomato, At (Arabidopsis thaliana) for Arabidopsis, Ca (Capsicum annuum) for pepper, Gm (Glycine max) for soybean, Os (Oryza sativa) for rice and Zm (Zea mays) for maize.

Collinearity Analysis of SlFWL Genes with Other Species
The extension and evolution of gene families in the genome are significantly influenced by tandem and fragmental replication between gene families, and tandem replication is a key mechanism for creating new copies of genes [28]. To investigate the evolutionary relationships of FWL genes in different plant species, we performed a synteny analysis of FWL genes between the genomes of tomato, Arabidopsis, rice (Oryza indica and Oryza japonica), maize, soybean and pepper ( Figure 5, Table S3). The results show that the number of collinear gene pairs between tomato and the other species ranged from 1 to 22, suggesting that these collinear genes might originate from the same ancestor and are func- software was applied to construct the phylogenetic tree with 1000 bootstrap replicates. The FWL proteins were used to run 1000 self-replicates and build a phylogenetic tree using the neighbor-joining method on the MEGA (Version 11) program. Based on branching, the FWL family proteins were classified into five subfamilies. Each subfamily is represented by a distinct color: purple for subfamily A, red for subfamily B, yellow for subfamily C, blue for subfamily D, and green for subfamily E. Sl (Solanum lycopersicum) stands for tomato, At (Arabidopsis thaliana) for Arabidopsis, Ca (Capsicum annuum) for pepper, Gm (Glycine max) for soybean, Os (Oryza sativa) for rice and Zm (Zea mays) for maize. and soybean genomes, while maize and rice had the lowest number of collinear genes. These findings indicate that FWL genes might have undergone a complex evolutionary history.

Expression Profile Analysis of SlFWL Genes
Expression profiles of SlFWL genes were analyzed using data from the public RNAseq database (http://ted.bti.cornell.edu/cgi-bin/TFGD/digital/home.cgi, accessed on 18 May 2022). The results show that each SlFWL gene exhibited a distinct tissue expression pattern ( Figure 6, Table S4). SlFWL2, SlFWL9, SlFWL11, SlFWL12 and SlFWL21 were abundantly expressed in roots, followed by hypocotyl, and had a comparatively low expression level throughout the fruiting stage. Except for SlFWL20, all SlFWL genes were expressed in roots. SlFWL1, SlFWL6, SlFWL7, SlFWL8, SlFWL13 and SlFWL20 were mainly expressed in cotyledons. SlFWL14, SlFWL15, SlFWL17 and SlFWL19 were specifically expressed in mature flowers. FW2.2, SlFWL3, SlFWL5, SlFWL10, SlFWL16 and SlFWL18 show different expression patterns in various tissues. The tissue-specific expression patterns of SlFWL genes suggest that they may be involved in different biological processes and have diverse functions. The abundant expression of SlFWL genes in roots indicates their potential role in root development and response to environmental stresses, which is consistent with the presence of stress-related elements in their promoter regions. The expression of SlFWL

Expression Profile Analysis of SlFWL Genes
Expression profiles of SlFWL genes were analyzed using data from the public RNA-seq database (http://ted.bti.cornell.edu/cgi-bin/TFGD/digital/home.cgi, accessed on 18 May 2022). The results show that each SlFWL gene exhibited a distinct tissue expression pattern ( Figure 6, Table S4). SlFWL2, SlFWL9, SlFWL11, SlFWL12 and SlFWL21 were abundantly expressed in roots, followed by hypocotyl, and had a comparatively low expression level throughout the fruiting stage. Except for SlFWL20, all SlFWL genes were expressed in roots. SlFWL1, SlFWL6, SlFWL7, SlFWL8, SlFWL13 and SlFWL20 were mainly expressed in cotyledons. SlFWL14, SlFWL15, SlFWL17 and SlFWL19 were specifically expressed in mature flowers.

Expression of SlFWL Genes in Response to Abiotic Stress
To investigate the function of the SlFWL gene family under abiotic stress, we examined the expression patterns of SlFWL genes in tomato leaves subjected to cold, heat, salt, and drought stresses at 0, 1, 3, 6, 12 and 24 h after stress treatments. FW2.2 and other SlFWL genes exhibited diverse expression patterns over time following various stress treatments, while SlFWL11, SlFWL15 and SlFWL17 were undetectable in plants under either control or abiotic stress treatments (Figure 7 and Figure S1-S4, Table S6).
Under cold stress, the majority of SlFWL genes were up-regulated. SlFWL2, SlFWL3, SlFWL6, SlFWL7, SlFWL8, SlFWL9, SlFWL12, SlFWL13, SlFWL16 and SlFWL19 exhibited a similar expression trend in response to cold stress, with a significant increase at 1 h of cold stress treatment, followed by a downward trend at 3 h or 6 h ( Figure 7A and Figure S1, Table S6). Notably, SlFWL2 increased by 28-fold in response to cold stress. However, the expression of SlFWL1 was remarkably different, showing a 150-fold increase in expression after 24 h of exposure to cold stress. Short-term cold stress treatment dramatically reduced the expression of FW2.2, SlFWL5, SlFWL10 and SlFWL14, after 12 h, their expression began to climb. Under cold stress, the expression of SlFWL18 and SlFWL21 constantly decreased. SlFWL20 did not respond to cold stress until 6 h, and its expression considerably increased by 10 times at 12 h.

Expression of SlFWL Genes in Response to Abiotic Stress
To investigate the function of the SlFWL gene family under abiotic stress, we examined the expression patterns of SlFWL genes in tomato leaves subjected to cold, heat, salt, and drought stresses at 0, 1, 3, 6, 12 and 24 h after stress treatments. FW2.2 and other SlFWL genes exhibited diverse expression patterns over time following various stress treatments, while SlFWL11, SlFWL15 and SlFWL17 were undetectable in plants under either control or abiotic stress treatments (Figure 7 and Figures S1-S4, Table S6).
Under cold stress, the majority of SlFWL genes were up-regulated. SlFWL2, SlFWL3, SlFWL6, SlFWL7, SlFWL8, SlFWL9, SlFWL12, SlFWL13, SlFWL16 and SlFWL19 exhibited a similar expression trend in response to cold stress, with a significant increase at 1 h of cold stress treatment, followed by a downward trend at 3 h or 6 h ( Figure 7A and Figure S1, Table S6). Notably, SlFWL2 increased by 28-fold in response to cold stress. However, the expression of SlFWL1 was remarkably different, showing a 150-fold increase in expression after 24 h of exposure to cold stress. Short-term cold stress treatment dramatically reduced the expression of FW2.2, SlFWL5, SlFWL10 and SlFWL14, after 12 h, their expression began to climb. Under cold stress, the expression of SlFWL18 and SlFWL21 constantly decreased. SlFWL20 did not respond to cold stress until 6 h, and its expression considerably increased by 10 times at 12 h.  Heat stress significantly altered the transcriptional profile of SlFWL genes ( Figure 7B and Figure S2, Table S6). SlFWL1, SlFWL2, SlFWL3, SlFWL5, SlFWL7, SlFWL8, SlFWL13, SlFWL16 and SlFWL21 exhibited a considerable increase in expression (1.25-59-fold) after 1 h of heat treatment in comparison to the control (0 h). Under heat stress, the expression of SlFWL1 instantly rose 59-fold at 1 h. SlFWL19 did not respond to heat stress before 6 h, and then reached its highest expression level. The response of SlFWL20 to heat stress lasted long and was consistently up-regulated within 24 h. The expression of SlFWL6, SlFWL9, SlFWL10 and SlFWL12 was down-regulated in the short term but peaked within 6-12 h under heat stress. FW2.2, SlFWL14 and SlFWL18 were down-regulated at 1 h under heat stress, and, after a brief recovery, their expression was significantly suppressed after 6 h.
In addition, most SlFWL genes responded to salt stress treatment ( Figure 7C and Figure S3, Table S6). Among these genes, SlFWL1, SlFWL2, SlFWL5, SlFWL6, SlFWL7, SlFWL8, SlFWL10, SlFWL12, SlFWL13, SlFWL16, SlFWL18, SlFWL19, SlFWL20 and SlFWL21 all exhibited varying degrees of increased expression. Within 1-12 h of salt stress treatment, SlFWL1, SlFWL2, SlFWL6, SlFWL7, SlFWL13 and SlFWL20 show substantial increases, with the expression level increasing more than 20-fold. Notably, SlFWL13 shows the most significant increase, with expression levels increasing more than 200-fold after 12 h of treatment. The expression of SlFWL3 and SlFWL9 reached its peak at 6 h of salt stress. However, FW2.2 and SlFWL14 were barely detectable after 24 h of salt stress treatment, indicating that salt stress inhibited their expression.
In summary, the study provides evidence for the differential expression of SlFWL genes under abiotic stress treatments. These findings could be helpful in developing strategies to improve the tolerance of crops to these environmental stresses.

Discussion
FWL proteins with the PLAC8 structural domain are known to be universally present in plants, mammals, and fungi and have been found to play critical roles in regulating plant organ size, metal ion homeostasis and root tumor formation [1,3,8,11]. Several FWL genes in different species have been identified, including GmFWL1, OsFWL3, ZmCNR3, MdCNR8, cell number regulators (CNRs) and Pafw2.2-like [3,8,11,17,29,30]. These FWL genes play a conserved role in repressing cell division or controlling cell number to control plant organ size [17]. However, it is yet unknown whether tomato FWLs react to abiotic stress. In this study, we performed a genome-wide identification of the tomato FWL (FW2.2-Like) genes and thoroughly analyzed the gene structure, chromosomal localization, phylogeny, gene duplication, cis-regulatory elements, expression profiles in different tissues and in response to different abiotic stress treatments in tomatoes.
In this study, we systematically identified a total of 21 SlFWL genes, including the well-known gene FW2.2, which is distributed on 10 chromosomes in tomatoes (Figure 1). Among them, SlFWL6 and SlFWL7, SlFWL11 and SlFWL12, SlFWL13 and SlFWL14, and SlFWL18 and SlFWL19 are connected two by two tandemly on ch03, ch06, ch08 and ch10, respectively. SlFWL genes exhibit a degree of conservation and segregation in physicochemical properties, such as amino acid length, molecular weight and isoelectric point. These SlFWL proteins range in size from 98 to 505 amino acids, but most are relatively short, between 100 and 300 amino acids. Each SlFWL protein contains an average of 6.7% Cys, and the conserved portion rich in cysteine is sometimes referred to as the PLAC8 motif (Table 1) [3].
Our anticipated subcellular localizations demonstrate that the SlFWLs, as well as FW2.2 and other FWL genes in other plants, are primarily distributed on the intracellular cell membrane. Compelling evidence suggests that FW2.2 interacts physically with the regulatory (beta) subunit of a CKII kinase at or near the plasma membrane. This discovery implied that FW2.2 may serve as a component of a signaling mechanism activated by extracellular signals that regulate fruit cell division [1,7]. Additionally, numerous studies have emphasized plant hormones and their interrelated roles in controlling fruit development and fruit size [31]. The tomato ovary consists of two or more carpels enclosing the locular containing the ovule. After successful fertilization, a period of cell division and cell expansion begins, which continues for 6-7 weeks [32]. During the period of cell division, the auxin concentration is likely to increase, some of which shows peak expression at the stage of cell expansion, suggesting that phytormone auxin plays a role in fruit initiation and fruit-size cell division by regulating cell division and cell expansion processes [33]. Cell proliferation and expansion are known to be regulated by intricate interactions between stimulatory signals, including hormone signaling and carbon partitioning through the activation of D-type cyclins [34]. For instance, FW2.2 may function in a signaling pathway that connects hormone or sugar signals to the control of cell cycle machinery in developing flowers. This pathway may affect inflorescence number by the modulation of photosynthate partitioning in the plant and may act upstream of several genes associated with cell proliferation, such as CYCD3; 1 and KRP [35]. However, whether FW2.2 is involved in the distribution and response of plant hormones is unknown, and the molecular mechanism by which FW2.2 connects hormone and sugar signals to regulate cell proliferation remains an open question.
The majority of members of the FWL family are transmembrane domain proteins, and several investigations have shown that they are frequently found localized in the plasma membrane of cells [14]. In other plants, AtPCR1 was localized at the plasma membrane [14]. The amino acid sequences of AtMCA1 and AtMCA2 share several common structural features, such as putative transmembrane (TM) segments and an EF hand-like region in the N-terminal half. These structural features are crucial for the activity of Ca 2+ uptake and can function as intact membrane proteins [24]. A TM fragment with the CC(L)XXXXCPC domain forms a pore with the hydrophilic side toward the lumen, cysteine residues are lined on the side toward the lumen, and metal ions can migrate through or interact with the channel [3]. Our transmembrane structure predictions show that more than half of the SlFWL proteins have at least 1 transmembrane structure with an N-terminal PLAC8 structural domain of CC(I/L/W/F/V)XXXXCPC with a change in the second amino acid C (Table S2). There are some SlFWL genes that do not contain this structure. We speculated that the function of these genes may have also changed during evolution, which remains to be further verified. It is worth mentioning that cadmium resistance can be achieved by solely expressing the AtPCR1 N-terminal hydrophobic segment, which carries a CCXXXXCPC sequence conserved among various organisms [14].
Differences in the functional expression of genes are inextricably linked to their structure [36]. The structural distribution characteristics of SlFWL genes are very similar, except for SlFWL9, which has no coding region. The position and distribution of the coding region CDS of all SlFWL genes are relatively conserved ( Figure 1A). Most of the members have 3-4 exons and are relatively evenly distributed. In contrast, SlFWL3 and SlFWL7 have longer genome sequences and more introns than the other genes, implying that they may be early FWL family members that achieve transcriptional diversification through processes such as selective splicing to regulate more complex and broad functions [37]. Further analysis of the conserved motif reveals that all SlFWL proteins have very high similarity, with most containing motif 1-5 and motif 10. Motif 3 and motif 4 are at the N-terminus, while motif 1 and motif 5 are distributed at the C-terminus (Figure 2). Motif 5 and motif 1 contain a CXXC sequence, which was exactly a cysteine-rich structural domain of unknown function, known as the PLAC8 or DUF614 motif [3,4,38]. Such structures flanked by cysteines are generally associated with the formation of redox-related proteins, isomerization, and a reduction of disulfide bonds [38]. This feature was consistent with the cysteine morphology of mammalian PLAC8, and this cysteine-rich PLAC8 structure is highly conserved in the SlFWL genes. We hypothesized that the PLAC8 structure of the SlFWL protein is also functionally conserved. Common motifs imply functional redundancy, while specific motifs may lead to functional divergence [39]. SlFWL13 and SlFWL20 do not contain motif 10, while motif 7 and motif 6 are unique to SlFWL5, 10 and 17, motif 8 and motif 9 are unique to SlFWL11 and SlFWL12. Pfam searches of the motifs revealed that motif 7 contains a zinc finger domain and an XPA protein N-terminal motif. It is clear that the distribution of SlFWL gene structures and conserved motifs forms different clusters, showing some evolutionary similarities and common functions, while the disappearance or appearance of additional motifs and structures may function in the expansion and diversity of the SlFWL gene family during evolution. The SlFWL proteins may have functionally diverged during evolution.
Gene duplication results in longer protein sequences, more functional domains, and more cis-regulatory motifs, and gene duplication increases the number of genes and the complexity of gene function [40]. We found a total of 68 associated FWL proteins in dicotyledonous plants (Arabidopsis, pepper and soybean) and monocotyledonous plants (rice and maize). These genes are distributed in five major branches (Figure 4). The results suggest that FWL is conserved in both monocots and dicots, implying the existence of a common ancestor. From an evolutionary perspective, gene duplication events increased the number of genes in specific gene families, thus enabling plants to adapt and survive under adverse environmental stresses [28,41]. Collinearity analysis revealed that the SlFWL genes are most closely related to the soybean genome and have the highest number (22 pairs) of colinear gene pairs between them, followed by pepper (17 pairs) ( Figure 5).
We analyzed 2000 bp of cis-regulatory elements (CREs) upstream of the translation start site of SlFWL family genes. The promoter region contains multiple light-responsive elements and hormone-related action elements such as abscisic acid-responsive elements, jasmonic acid-responsive elements, gibberellin-responsive elements, and salicylic acidresponsive elements. In addition, these SlFWL genes also have stress-signaling elements such as TC-rich MYB-and MYC-binding sites (Figure 4). These results show that SlFWL expression is closely associated with abiotic stress and hormone signaling responses. Earlier studies identified a conserved cis-acting regulatory element in the promoter of droughtinducible genes called the ABA response element (ABRE) [42]. MYB proteins are key factors in the regulatory network controlling development, metabolism, and responses to biotic and abiotic stresses, and several stress-related MYB elements have been reported in stress resistance gene promoters [43,44]. Our findings reveal that the majority of SlFWL members feature MYB-binding elements. Additionally, the SlFWL gene family encompasses the GCN4 motif, which is developmentally significant and appears in numerous seed storage protein genes. Studies have demonstrated that promoter fragments carrying the GCN4 motif serve to regulate the seed-specific expression of genes related to germination [45]. The cis-acting elements suggested that the SlFWL genes may play an important role in light response, phytohormone signaling, and stress response.
The expression pattern of FWL genes in different tissues has been described in many species. For example, in Arabidopsis, reverse transcription PCR (RT-PCR) analysis shows that MCA2 was expressed in leaves, flowers, roots, angiosperms and stems, the GUS reporter gene for MCA1 was expressed in cotyledons, leaves, vascular tissues of the main roots and regions of the rosette center corresponding to the stem apical meristem [27]. However, SlFWL does not have a uniform gene expression pattern in tomatoes. We obtained each SlFWL gene expression pattern in different tissues from public databases ( Figure 6, Table  S4). Ten SlFWL genes were differentially expressed in all tissues of the whole root (ROOT), hypocotyl (HYPO), nutritional meristem (MERI), cotyledon (COTYL), young leaf (YL), mature leaf (ML), young bud (YFB), flower (0 DPA), 10 days post-anthesis fruit (10 DPA), 20 days post-anthesis fruit (20 DPA) and ripe fruit (33 DPA). SlFWL1, 2, 3, 5, 10, 12, 16 and 19 were highly expressed in all tissues, with higher expression than other SlFWL genes.
Expression clustering analysis revealed that SlFWL2, 9, 11, 12 and 21 were highly expressed in roots, followed by hypocotyl, and were relatively low in the fruiting stage. SlFWL1, 6,7,8,13 and 20 were more highly expressed in cotyledons and roots than in any other tissues, and SlFWL14, 15, 17 and 19 were specifically expressed in mature flowers. The differential expression of these SlFWL genes in different tissues may indicate that they perform different functions in different parts of the plant and remains to be further investigated.
Unfavorable abiotic stress environmental conditions, such as cold, heat, drought and excess salt in the soil, or toxic metals such as aluminum, arsenic, cadmium, etc., are often detrimental or stressful to growth and development, while drought, salt and temperature stress are the main environmental factors that limit the productivity of agricultural plants and threaten food security [46]. Analysis of gene expression patterns during growth and development and exposure to stress stimuli may help determine their functions. Currently, the functions of members of the FWL family are focused on fruit and whole plant size and heavy metal resistance and allocation, but roles in abiotic stresses have not been reported [22,47]. In our study, we examined the transcriptional profiles of the SlFWL family under cold temperature, high temperature, salt, and drought treatments (Figure 7 and Figure S1-S4, Table S6). SlFWL11, 15 and 17 were not detectable in their expression in leaves under either control or abiotic stress treatments, which was consistent with the data from RNA-seq (Table S4). Gene structure analysis revealed that SlFWL9 is a pseudogene that is able to respond to different stress treatments despite the absence of exons in its gene structure. FW2.2 was repressed by all stresses, and its expression was significantly reduced under all abiotic stresses. SlFWL1, 2, 3, 7, 8, 13, 16 and 19 were significantly upregulated under both cold and heat stress treatments. SlFWL10, 14 and 18 were significantly down-regulated under cold and high temperature stresses. SlFWL5, 6, 12, 20 and 21 show opposite trends in cold and high temperature stress expression trends. SlFWL1, 3, 6, 7, 8, 9, 10, 12, 13, 14, 19, 20 and 21 show similar expression profiles under drought conditions with those under salt stress, with a simultaneous increase or decrease in the short term. These studies suggested that members of the SlFWL gene family may have different functions in response to different environmental stimuli, and further studies on their role in response to abiotic stress are needed in the future.
The results of the expression profiles of the SlFWL genes as well as in response to salt, drought, cold and heat stress treatments reveal that although some SlFWL genes are constitutively expressed in various tissues, they are stress-inducible, suggesting that they play multiple roles in different tissues and in response to different abiotic stress conditions. For instance, SlFWL8 and SlFWL9 were upregulated in response to salt stress, while SlFWL5 and SlFWL15 were upregulated in response to drought stress. Some SlFWL genes show tissue-specific expression patterns, such as SlFWL1, which was highly expressed in the root and flower, and SlFWL2, which was highly expressed in the fruit. These findings suggest that the SlFWL genes may have tissue-specific and stress-responsive functions in tomatoes.

Plant Materials and Growth Conditions
The study was performed on tomato cultivar NR21-16, kept in our laboratory. Tomato seedlings were grown in a growth chamber at 25 • C/16 h and 20 • C/8 h photoperiods (light/dark) with a light intensity of 300 µmol·m −2 s −1 and 60-70% humidity [48].
To investigate the expression pattern of the SlFWL genes under different abiotic stress conditions (cold, heat, salt [NaCl], and drought [PEG]), we subjected 30-day-old (four-leafstage) tomato plants with relatively uniform growth conditions to stress treatments for three days and collected leaf samples at 0, 1, 3, 6, 12, and 24 h after the onset of stress [49]. Plants were incubated in growth chambers at 4 • C and 42 • C to apply cold and heat stress, respectively. Salt stress was applied by submerging the roots in a 200 mM NaCl solution [49]. For the drought treatment, PEG6000 was used to simulate drought, and the final concentration of PEG6000 in the hydroponic medium was 12%, and the roots needed to be fully submerged in the PEG6000 solution [50]. The third completely developed leaf of the treatment was collected. Plants with three independent biological seedlings in good condition were used for each sample. Leaf tissues from each biological replicate were collected and mixed thoroughly, then immediately frozen in liquid nitrogen and stored at -80 • C for RNA extraction.

RNA Extraction and Expression Analysis
Total RNA from tomato leaves was extracted using the Biospin Plant Total RNA Extraction Kit (Hangzhou Borui Technology Company, Hangzhou, China) and then reverse transcribed to obtain cDNA by the HiScript II One Step RT-PCR Kit [51]. Quantitative realtime PCR was performed with Hieff UNICON ® Universal Blue qPCR SYBR Green Master Mix reagent (Yessen Biotechnology Co., Shanghai, China), and primers were designed by NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 8 August 2022) ( Table S5). The expression level of the tomato eukaryotic initiation factor gene (eiF, Solyc12g096000) was used as an internal control. The relative expression levels of the genes of interest were calculated using the 2 -∆∆CT method. Each reaction was performed in three replicates.

Phylogenetic Relationship, Gene Structure, Protein Motif and Cis-Regulatory Element Analysis of the SlFWL Gene Family
All identified SlFWL genes in tomatoes were aligned using ClustalW, and the phylogenetic tree of SlFWL genes was generated using the neighbor-joining (NJ) method, the 1000 bootstrap method, and the Poisson model on MEGA (Version 11) [59]. The exonintron gene structures of the SlFWL genes were visualized by TBtools (Gene Structure Visualization (from GTF/GFF3 File)) [53]. The conserved structural domain was analyzed in the Pfam database. Conserved motifs were predicted by the MEME Suite tools (http://meme-suite.org, accessed on 16 May 2022), and the number of motif parameters was manually limited to 10 [60]. The promoter regions (2000 bp upstream of ATG) of the SlFWL genes were extracted from the tomato genome sequence using TBtools (v1.09876) software [53]. The promoter sequences were submitted to the PlantCARE online database for analysis (http://bioinformatics.psb.ugent, accessed on 12 May 2022), and these cisregulatory elements were drawn by TBtools (v1.09876) [53,61].

Evolutionary Relationships and Synteny Analysis of SlFWL Genes in Multiple Species
Multiple sequence alignment analyses of the FWL homologous family members in Arabidopsis thaliana, rice, maize, pepper and soybean were performed using the MUSCLE alignment function in the MEGA (Version 11) with default settings [59]. The maximumlikelihood method of the IQ-TREE function in the TBtools (v1.09876) software was applied to construct the phylogenetic tree with 1000 bootstrap replicates [53]. Then, the data were visualized and optimized with the online tool Interactive Tree of Life (iTOL) (https: //itol.embl.de/, accessed on 26 May 2022) to generate a phylogenetic tree [62]. The collinearity analysis was performed with One Step MCScanX of the TBtools (v1.09876) software [53].

Tissue Expression Pattern of the SlFWL Gene Family
We obtained tissue expression data of the wild tomato variety LA1589 from the publicly available transcriptome database, Tomato Functional Genomics Database (http: //ted.bti.cornell.edu, accessed on 18 May 2022), which included the whole root (ROOT), hypocotyl (HYPO), vegetative meristems (MERI), cotyledons (COTYL), young leaves (YL), mature leaves (ML), young flower buds (YFB), anthesis flowers (0 DPA), 10 days postanthesis fruit (10 DPA), 20 days post anthesis fruit (20 DPA), and ripening fruit (33 DPA) [63]. We retrieved the FPKM (fragments per kilobase per million reads) values representing the expression levels of SlFWL genes. The data for each row were normalized and plotted according to the average FPKM value for each gene. Heatmaps were generated using R software (v.4.2.2), and the package "pheatmap" (v.1.0.12) through Hiplot, a comprehensive web service for biomedical data analysis and visualization [64]. The qRT-PCR data for abiotic stresses were also used for heatmap analysis.

Statistical Analysis
Data was collated using Microsoft Excel 2016 and analyzed for statistics and significance using SPSS software 20.0 (IBM, Armonk, NY, USA), with one-way ANOVA and Duncan's test (p < 0.05) indicating a significant difference. Drawing was conducted using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA).

Conclusions
The FW2.2-like (FWL) gene family in tomatoes, including gene structure, chromosomal location, phylogeny, gene duplication, cis-regulatory elements and expression patterns in response to various abiotic stress treatments, is thoroughly analyzed in this paper. These findings imply that SlFWL genes may serve a variety of abiotic stress-related purposes. In addition to facilitating future functional research of this gene family in tomatoes and other plant species, the discovery and characterization of SlFWL genes in this work may have repercussions for the future creation of stress-tolerant crops.

Conflicts of Interest:
The authors declare no conflict of interest.Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.